Two general types of polynucleotide diagnostic systems, both based on hybridization between complementary segments of a polynucleotide probe and a single-strand polynucleotide analyte, have been developed. In the first type, the polynucleotide analyte is made single stranded and fixed to a solid support, such as a nitrocellulose filter. The support is then reacted, under complementary-strand annealing conditions, with a reporter-labeled probe which is complementary to a target base-sequence region of the analyte. After washing several times to remove unbound probe, the solid support is analyzed for the presence of reporter. This system has not been entirely satisfactory, particularly in clinical situations, where assay simplicity and sensitivity are required. The procedure used in fixing single-strand polynucleotide material to the solid support is somewhat involved and time consuming. The sensitivity of the system is limited because, in the usual case, each analyte molecule hybridizes with a single probe molecule and each probe generally contains no more than a few dozen reporter moieties.
A second type of polynucleotide diagnostic system involves two analyte-specific probes which are each complementary to a distinct region of the analyte polynucleotide. The first probe is linked to a solid support, and the second probe is free in solution and carries multiple reporter molecules. In practice, the analyte is mixed with the two probes under conditions which allow annealing of complementary polynucleotide strands, including annealing of both the immobilized and reporter-carrying probes to the polynucleotide analyte, to attach the reporter to the solid support by means of the analyte.
Although the dual-probe system avoids the problem of having to fix the test nucleic acid material to a solid support, nevertheless, the method has a number of limitations. First, when the test nucleic acid is derived from duplex nucleic acid, as is often the case, hybridization between the analyte polynucleotide and its complementary strand competes with the hybridization between the analyte and the two probes. Further, since the two-probe system relies on higher order kinetics than is the case for single-probe systems, it is inherently slower than a single-probe system. Also, the need for two different probes increases the cost of the system. Finally, in terms of test sensitivity, the dual-probe system suffers the same limitation as the single-probe system mentioned above--each analyte polynucleotide binds only one "reporter" probe.